Overactivity of glycogen synthase kinase 3 (GSK-3) is associated with insulin resistance of skeletal muscle glucose transport in prediabetic and type 2 diabetic rodent models. However, limited information is available concerning the potential molecular mechanisms underlying the role GSK-3 plays in the etiology of insulin resistance in the male Zucker Diabetic Fatty (ZDF) rat, a model of type 2 diabetes mellitus. Therefore, we assessed the functionality of proximal and distal insulin signaling elements in isolated type I (slow-twitch oxidative) soleus muscles of ZDF rats after in vitro exposure to a selective GSK-3 inhibitor (1 μmol/L CT98014, K_i <10 nmol/L for GSK-3α and GSK-3β). Moreover, Ser307 phosphorylation of insulin receptor substrate 1 (IRS-1), which has been implicated in the development of insulin resistance, was also determined in the absence or presence of this GSK-3 inhibitor. Maximally insulin-stimulated (5 mU/mL) GSK-3β serine phosphorylation was significantly less (35%, P < .05) in soleus muscle of ZDF rats compared with insulin-sensitive lean Zucker rats, indicating GSK-3 overactivity. In the absence of insulin, no effects of GSK-3 inhibition were detected. GSK-3 inhibition led to significant enhancement (28%) of insulin-stimulated glucose transport activity that was associated with significant up-regulation of tyrosine phosphorylation of IR (52%) and IRS-1 (50%), and with enhanced Akt Ser473 phosphorylation (48%) and GSK-3β Ser9 phosphorylation (36%). Moreover, the selective GSK-3 inhibitor induced a significant reduction in the phosphorylation of IRS-1 Ser307 (26%) and c-jun N-terminal kinases 1 and 2 (31%), a mediator of IRS-1 Ser307 phosphorylation. These results indicate that selective inhibition of GSK-3 activity in type I skeletal muscle from overtly diabetic ZDF rats enhances IRS-1–dependent insulin signaling, possibly by a decrease in c-jun N-terminal kinase activation and a diminution of the deleterious effects of IRS-1 Ser307 phosphorylation.